(T1030-11-60) Challenges with Removal and Addition of Polysorbates From/To Formulations of Monoclonal Antibodies

Purpose: Membrane separation techniques, including ultrafiltration and dialysis, are crucial in the development and manufacturing of protein products. These methods are extensively used for tasks such as buffer exchange, desalting, and adjusting protein concentrations, playing a pivotal role in both removing unwanted components and transferring proteins to suitable analysis buffers. They are essential in formulating therapeutic proteins, excipient variations, conducting stability and compatibility assessments during early pharmaceutical development. Additionally, these techniques are vital for preparing samples for analytical assessments such as Mass spectroscopy, and for biosimilar analyses to ensure appropriate buffer conditions for downstream processes. While large-scale ultrafiltration/diafiltration is common in manufacturing, small laboratories often rely on less understood small-scale techniques. This study compares the effectiveness of centrifugal concentrators with a two-step approach involving a surfactant removal column for polysorbate elimination. Moreover, a comparison between two different approaches of polysorbate addition in formulation was tested.

Methods: Studying polysorbate behavior and Tmab stability in centrifugal concentrators - Centrifugal concentrators with 30kDa membrane cutoff were used for buffer exchange of Trastuzumab biosimilar (Tmab) formulations. Initially, 15mg of Tmab (1.5ml at 10mg/ml) from Herzuma commercial formulation underwent four buffer exchange cycles with histidine buffer, each cycle involving 2.5 minutes of centrifugation at 2000xg, to remove polysorbate. Polysorbate levels were measured before and after the process using a modified fluorescence assay method (Zheng et al). We established a standard curve with polysorbate 20 concentrations ranging from 0.001% to 0.01%, in the presence of 10mg/mL Tmab, using BIS-ANS as the fluorescent probe. In a separate experiment, reformulation of Tmab was performed using centrifugal concentrators to study polysorbate 20 behavior when it is present in the formulation buffer. Starting with 7mg of Tmab (0.7ml at 10 mg/ml), the buffer containing trehalose and polysorbate 20 in histidine buffer, as per the composition of Herzuma® product, was exchanged in four cycles. Post-exchange, Polysorbate 20 concentration was measured and Tmab stability was assessed using size exclusion chromatography (SEC), Dynamic light scattering (DLS), Flow Imaging Microscopy (FlowCam), Far-UV Circular dichroism (CD), UV, and fluorescence spectroscopy (FLU).Studying polysorbate behavior and Tmab stability in surfactant removal columns - The surfactant removal column, designed to extract polysorbate from protein solutions efficiently, contains a resin with high polysorbate binding capacity. Tmab formulation was first incubated in the column for 30 minutes and the flow-through sample was collected. Subsequently, four buffer exchange cycles with histidine buffer were performed to eliminate trehalose and yield Tmab in histidine buffer. The effectiveness of polysorbate removal and the stability of Tmab was confirmed using the same analytical techniques mentioned previously. To ensure optimum reformulation of Tmab, we propose an approach in which the polysorbate will be added separately after completing the buffer exchange step using a buffer containing only trehalose in histidine buffer.

Results: Efficiency of centrifugal concentrators alone vs surfactant removal column followed by buffer exchange for removal of polysorbates. Initial measurements show the polysorbate concentration in Herzuma® to be 0.022±0.003mg/ml. Using a centrifugal concentrator with a 30kDa membrane cut-off, polysorbate levels are found to be 0.018±0.002mg/ml after four buffer exchange cycles. In contrast, the surfactant removal column eliminated polysorbate. SEC analysis shows insignificant differences in the high molecular weight percentage (values below 1%) while in DLS, there is a slight increase in particle diameter of Tmab processed by surfactant removal column (figure 1, a-b). CD and UV-2^nd derivative, FLU analysis revealed minor structural alterations without significant impact on protein structure (figure 2). Implications of Polysorbate Content in Centrifugal Concentrator Buffer Exchange - After reformulation using a 30kDa centrifugal concentrator and four buffer exchange cycles with polysorbate present in the formulation buffer, polysorbate concentration significantly increased tenfold to 0.23±0.1mg/ml (p-value < 0.001), indicating a tendency for polysorbate to concentrate using membrane separating techniques. SEC and DLS showed insignificant differences in HMW% (less than 1%) or hydrodynamic particle size (around 10nm) between different formulations, respectively (figure 1, c-d). FlowCam analysis revealed a significant rise in particle count in Tmab formulated through the two steps approach (figure 1, e). CD spectra indicated higher ellipticity at 218nm in Tmab after reformulation using buffer exchange (figure 3, a), suggesting a disruption in secondary structure particularly where polysorbate accumulated. Intrinsic fluorescence and UV-2^nd derivative analyses showed insignificant changes in protein structure.

Conclusion: Tmab does not show significant instability or aggregation during isolation. Buffer exchange using centrifugal concentrators where formulation buffer contains polysorbate led to accumulation of polysorbate in the samples. A solution to this problem was applied by adding polysorbate in a separate step after buffer exchange (two-step approach). The selection and quantity of polysorbate incorporated into the formulation of biotherapeutics should not be arbitrary; rather, it must be based on empirical data and the decision regarding polysorbate usage should be tailored to individual characteristics. This insight is particularly valuable for small companies engaged in biologics formulation as they lack formulation platforms akin to big pharmaceutical companies.

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Acknowledgements: This project is supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services (HHS) as part of a financial assistance award U01FD007758 to the National Institute for Pharmaceutical Technology and education (NIPTE) totaling $1,976,692 with 100 percent funded by FDA/HHS. The contents are those of the author(s) and do not necessarily represent the official views of, nor an endorsement, by FDA/HHS, or the U.S. Government.


Figure 1: (a) HMW% of Tmab processed by centrifugal concentrator and Surfactant removal column vs Tmab of Herzuma® formulation as a control (before isolation). The figure shows insignificant difference in HMW% of Tmab samples. (b) Particle hydrodynamic diameter of Tmab processed by centrifugal concentrator and surfactant removal column vs Tmab of Herzuma® formulation as a control (before isolation). The figure shows insignificant difference in diameter of Tmab samples. (c) HMW% of Tmab formulated using two different approaches, compared to Tmab in Herzuma® formulation as a control. The figure shows insignificant difference in HMW%. (d) Particle hydrodynamic diameter of Tmab formulated using two different approaches, compared to Tmab in Herzuma® formulation as a control showing insignificant difference. (e) Particle count per ml of Tmab formulated using two different approaches, compared to Tmab in Herzuma® formulation as a control (before isolation). The figure shows a statistically significant rise in particle count in Tmab formulated through the two steps approach.


Figure 2 (a) Secondary structural transition during Tmab processed by centrifugal concentrators (red) and Tmab processed by Surfactant removal column (black), (b) Fluorescence signal of Tmab processed by centrifugal concentrator and Surfactant removal column vs Tmab of Herzuma® formulation as a control (before isolation) showing no peak shift. (c) UV Second derivative of Tmab processed by centrifugal concentrator and Surfactant removal column vs Tmab of Herzuma® formulation as a control (before isolation) showing some changes in the signal around 280 nm, corresponding to tryptophan residues.


Figure 3: (a) The secondary structure stability of Tmab formulation by buffer exchange with polysorbate in formulation buffer (magenta) and Tmab formulation by buffer exchange then polysorbate addition (green) where the former contains high polysorbate content. (b) Figure 5: Fluorescence signal of Tmab formulated using two different approaches, compared to Tmab in Herzuma® formulation as a control (before isolation) showing no peak shift. (c) UV second derivative of Tmab formulated using two different approaches, compared to Tmab in Herzuma® formulation as a control (before isolation). showing some changes in the signal around 280 nm, corresponding to tryptophan residues.